Screening of differentially expressed genes in human renal cell carcinoma using suppression subtractive hybridization.
- Author:
Ying WANG
1
;
Wei CHEN
;
Xu LI
Author Information
- Publication Type:Journal Article
- MeSH: Carcinoma, Renal Cell; genetics; pathology; Cell Line; Cell Line, Tumor; Gene Expression Profiling; methods; Gene Expression Regulation, Neoplastic; Humans; Kidney Neoplasms; genetics; pathology; Nucleic Acid Hybridization; methods
- From: Journal of Southern Medical University 2008;28(1):89-93
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVETo suppress COL1A1 and COL3A1 gene expressions in human skin fibroblasts (HSFs) by means of RNA interference (RNAi).
METHODSSSH was performed in two directions to isolate the differentially expressed genes between human a RCC cell line RLC-310 and a normal renal cell line HK-2 (ATCC). The cDNAs obtained from the final nested PCR were directly inserted into T/A cloning vector to establish a subtractive cDNA library of specifically or highly expressed genes in RCC. Reverse Northern dot blotting was performed to screen the truly differentially expressed genes, and 200 positive genes were randomly selected for sequencing.
RESULTSThe two-directional subtractive libraries contained more than 1200 clones, and 213 positive clones were obtained using reverse Northern blotting. Sequence analysis of these clones identified 144 differentially expressed genes, including 67 up-regulated and 77 down-regulated genes, in which 14 novel ESTs and 21 functionally unknown genes were found. Cluster analysis indicated the involvement of the sequenced genes in cell growth, cell adhesion and apoptosis.
CONCLUSIONReliable subtractive cDNA libraries of human RCC have been constructed successfully with SSH. The identification of the gene expression profile in RCC may help clarify the mechanism of tumorigenesis and development of RCC, and also sheds light on new targets for prevention, diagnosis and therapy of this malignancy.
